Compositions and Methods for the Systemic Treatment of Arthritis

ABSTRACT

The present invention includes compositions and methods for treating arthritic joints found in patients with autoinflammation, e.g., systemic onset juvenile idiopathic arthritis, by administering at the site of inflammation a therapeutically effective amount of at least one agent that reduces or blocks the bioavailability of interleukin-1β.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60,548,033, filed Feb. 26, 2004, the entire content of which isincorporated herein by reference.

TECHNICAL FIELD OF INVENTION

The present invention relates to therapeutic use of a bio-affecting andbody-treating composition, and more particularly, to compositions andmethods for the systemic treatment of joint inflammation.

BACKGROUND OF THE INVENTION

Juvenile idiopathic arthritis (“JIA”), also referred to as juvenilerheumatoid arthritis (“JRA”) or juvenile chronic arthritis (“JCA”) isthe most common rheumatic disease in childhood and an important cause ofboth short and long-term disability. The term “JIA” includes aheterogeneous group of diseases, each of which may have a variety ofcauses and elicit a variety of host responses. All are characterized,however, by the development of idiopathic peripheral arthritis thoughtto be secondary to an immuno-inflammatory pathogenesis, possiblytriggered by contact with external antigen(s). JIA is classifiedaccording to three major types of disease presentation: (1)oligoarthritis; (2) polyarthritis; and (3) systemic onset juvenileidiopathic arthritis (hereinafter “SOJIA”). Each of these groups isdefined by a constellation of clinical signs and symptoms during thefirst six months of illness.

Of the known JIA diseases, all three major groups based on primary modesof onset have been considered to: (1) have an “autoimmune etiology”; and(2) follow a complex, nonmendelian, genetic trait. Evidence ofunderlying autoimmunity is mainly drawn from the presence ofautoantibodies and/or immune complexes in the patient's sera.Anti-nuclear antibodies occur in a high proportion of children witholigoarticular and, to a lesser extent, polyarticular onset, and theseantibodies confer a higher risk of developing uveitis. Otherautoantibodies directed against cytoplasmic and extracellular componentssuch as collagen have also been described. Rheumatoid factors (RF) aredetected in approximately 3% of children with JIA, and the presence ofthese autoantibodies defines a subgroup of polyarticular onset patientswhose disease is undistinguishable from adult onset rheumatoid arthritis(Cassidy, J T and Ross, E. 2001. Textbook of Pediatric Rheumatology, 4thed, Books on Demand Publishers, Visby, Sweden; p. 218-321).

A variety of T cell alterations have been described mainly in the bloodand synovial fluid of children with oligoarthritis, but a consistentpattern of cytokine production has not been found. In botholigoarthritis and polyarthritis patients, increased soluble IL-2R andIL-1β have been found in the blood. In all three forms of JIA, TNF-α andsoluble TNFαR (p55/75) have been found elevated in the serum and/orsynovial fluid (SF). In SOJIA patients, abnormal expression of IL-6 hasbeen described, especially in the blood prior to the febrile spikes, andIL-6 levels correlate with the systemic activity of the disease, withthe development of arthritis, and with the increase in acute phasereactants (Yokata, S. 2003. “Interleukin 6 as a therapeutic target insystemic-onset juvenile idiopathic arthritis,” Curr Opin Rheumatol15:581-586). IL-6 deregulation has also been proposed to mediate thelinear growth retardation, thrombocytosis and anemia seen in SOJIApatients (de Benedetti F, et al., 1991. Arthritis Rheum 34:1158-1163;Mangge H, et al. 1999. J Interferon Cytokine Res 19:1005-1010; Ozen S,et al. 1997. Clin Rheumatol 16:173-178; de Benedetti F, et al., 1999. JRheumatol 26:425-431; Muller K, et al. 1998. Br J Rheumatol 37:562-569;Shahin A, et al., 2002. Rheumatol Int 22:84-88; de Benedetti F, et al.,1992. Clin Exp Rheumatol 10:493-498; and Muzaffer M, et al., 2002. JRheumatol 29:1071-1078).

Data on HLA segregation suggests a hereditary basis to the complexetiopathogenesis of the disease(s) in cases of oligoarticular andpolyarticular onset JIA. Studies in SOJIA have yielded, however,inconsistent results. Finally, both viruses and bacteria have beendescribed in association with chronic arthritis in children. Rubella andParvovirus B19 can cause chronic arthritis secondary to persistence ofthe organism or by triggering an immune reaction, as it has also beendescribed in cases of post-vaccination arthritis.

SOJIA represents about 10% of all the cases of JIA. The course andprognosis of SOJIA is heterogeneous, as fifty per cent of patients havea monophasic course with resolution of the symptoms, while the remainingfifty per cent develop a chronic relapsing and remitting course and avery severe form of polyarticular chronic arthritis. Patients with SOJIAalso display an increased risk of developing hemophagocytic syndrome, apotentially fatal complication (Cassidy, J T and Ross, E. 2001. Textbookof Pediatric Rheumatology, 4th ed, Books on Demand Publishers, Visby,Sweden; p. 218-321).

Children with SOJIA present with severe systemic symptoms (fever andrash) that usually precede the development of arthritis for weeks toyears. The high spiking fever, which is the hallmark of this disease,usually follows a quotidian pattern with 1-2 spikes/day. Patients lookcharacteristically well when the fever is not present, but become quiteill with the spikes. In many patients, the fever is accompanied by asalmon-pink rash that becomes more apparent with the fever.Additionally, children with SOJIA may have hepatosplenomegaly,lymphadenopathy, pericarditis and other manifestations of serositis.These systemic manifestations may last from weeks to months andeventually tend to subside to be followed by the development of chronicarthritis. About 50% of patients will present oligoarticular involvementand will eventually recover. The other half will evolve into apolyarticular pattern, the prognosis of which correlates with the numberof joints involved six months into the disease course. Up to 48% ofchildren with SOJIA will have active arthritis ten years after thediagnosis is made (Cassidy, J T and Ross, E. 2001. Textbook of PediatricRheumatology, 4th ed, Books on Demand Publishers, Visby, Sweden; p.218-321; and Lomater C, et al., 2000. J Rheumatol 27:491-496).

There are no available specific tests to establish the diagnosis ofSOJIA, nor are there known prognostic indicators to ascertain itsclinical course. Fever, anemia, leukocytosis and elevated erythrocytesedimentation rate (ESR) are the main initial features of the disease,sometimes lasting several months before the diagnosis can beestablished. As these symptoms are nonspecific and can mimic infections,malignancies, and other diseases, patients undergo a series of verycostly diagnostic tests and prolonged hospitalizations.

One of the most serious complications in patients with SOJIA is thedevelopment of hemophagocytic syndrome, also known as macrophageactivation syndrome (MAS) (Cassidy, J T and Ross, E. 2001. Textbook ofPediatric Rheumatology, 4th ed, Books on Demand Publishers, Visby,Sweden; p. 218-321). The hemophagocytic syndrome, which can occur aswell in the context of infectious and neoplastic diseases, is associatedwith serious morbidity and/or death. Its etiology, especially in thecontext of SOJIA, is unknown. Familial cases of MAS occur as the resultof defective viral killing due to mutations in genes like perforin(involved in the release of granzyme by cytotoxic T cells/natural killercells to target cells) or Rab27 (involved in the control of granzymevesicle degranulation).

SUMMARY OF THE INVENTION

Therefore, SOJIA remains a chronic inflammatory disease of unknownetiology for which a specific treatment has not been determined. Despiteextensive study, multi-drug treatment of patients with SOJIA is similarto that of oligoarthritis and polyarthritis, which depends on the phase(systemic phase versus arthritic phase) of the disease and on the extentof involvement. While a minority of patients does well withnon-steroidal anti-inflammatory drugs (NSAIDs), most children requirethe use of oral and/or parenteral steroids as well as methotrexate tocontrol the symptoms. Intravenous immunoglobulin (IVIG) has been used inrecalcitrant cases. Most recently, anti-TNF therapy (e.g., etanerceptand infliximab) is being added to these regimens. Nevertheless, thereare some limitations and risks associated with these drugs. For example,long term treatment of these patients with immunosuppressive drugs suchas methotrexate and steroids have been reportedly associated with animpaired ability of the patients to eliminate viral infections.Intravenous immunoglobulin treatment is accompanied by the risksassociated with transfusions of blood-derived products. The presentinventors have found that SOJIA patients do not respond as well andrequire higher doses of anti-TNF agents to control the symptoms than anyother type of JIA. Therefore, there is a continuing need to identifyeffective drug treatments for SOJIA disease.

Blocking IL-1 has been reported as an effective agent for treatingpatients with inflammatory arthritis associated with autoimmunediseases. Clinical studies have been preformed on adults with rheumatoidarthritis and children with diverse forms of juvenile arthritis.Anakinra (Kineret®, made by Amgen), the recombinant human IL-1 receptorantagonist has the ability to block the interaction of IL-1 with itsreceptor, thus blocking the cellular response of IL-1. A clinical trialusing Anakinra has been conducted with patients with severe rheumatoidarthritis. At 150 mg per day, a 43% response rate based on the AmericanCollege of Rheumatology index was seen in comparison to 27% in placebogroup. Clinical studies using IL-1 antagonists, however, have never beendone in children or adults with Systemic Onset Arthritis during thesystemic phase of the disease, as defined by the presence of feverand/or rash with or without arthritis. Furthermore, IL-1 has beenreported not to play a relevant role in SOJIA disease (de Benedetti F,et al., 1995. “Circulating levels of interleukin 1 beta and ofinterleukin 1 receptor antagonist in systemic juvenile chronicarthritis,” Clin Exp Rheumatol 13:779-84).

Autoinflammatory diseases are illnesses associated with alterations ofthe innate immune system. Cells of the innate immune system, e.g.,neutrophils, monocytes, macrophages and NK cells, are characterized bythe lack of somatically generated receptors, the clonal expansion ofantigen-specific cells and their inability to confer “immunologicalmemory.” Although autoinflammatory diseases may clinically mimicautoimmune diseases, patients with autoinflammation never developautoantibodies, which are the hallmark of many autoimmune diseases. Onthe other hand, most autoinflammatory diseases cause fever, usually withan intermittent or periodic pattern, and the skin and joints arefrequently the targets of inflammation. In the past five years, thegenes responsible for at least nine familial autoinflammatory diseaseshave been described. These include Familial Mediterranean Fever (FMF),Tumor Necrosis Factor-Associated Periodic Fever Syndrome (TRAPS), HyperIgD, Cyclic Neutropenia, Familial Cold Urticaria (FCU), Muckle-WellsSyndrome, Chronic Inflammatory Neurologic and Articular Syndrome(CINCA), Familial Granulomatous Disease (Blau's Syndrome), and Crohn'sdisease (reviewed in Hull K M, et al., 2003. Curr Opin Rheumatol15:61-69). Although most autoinflammatory diseases are the result ofsingle gene mutations, there are some that follow complex genetictraits, like ulcerative colitis and some forms of Crohn's disease, andyet some, like Behcet's, that may not be genetic in origin.

Despite reports the contrary, the present inventors have found thatSOJIA is an autoinflammatory disease and reducing or blocking thebioavailability of interleukin-1 beta (IL-1β) is an effectivetherapeutic treatment for SOJIA patients. In one aspect, the presentinvention is a method for treating a patient with systemic onsetjuvenile idiopathic arthritis by administering to the patient acomposition with an effective amount of at least one agent capable ofreducing or blocking the bioavailability of interleukin-1 beta.

More particularly, the present invention includes compositions andmethod for treating a joint undergoing autoinflammation, e.g., havingsystemic onset juvenile idiopathic arthritis, by administering to thejoint a composition that includes a therapeutically effective amount ofone or more active agents that reduces the bioavailability ofinterleukin-1β. The active agent may modify the transcription of theinterleukin-1β gene or the translation of the interleukin-1β gene.Alternatively, the active agent may be a single-stranded RNA molecule, adouble-stranded RNA molecule, an antisense RNA molecule, a smallinhibitory RNA (siRNA) molecule, or an inhibitory RNA molecule (RNAi)designed to reduce the expression of interleukin-1β. Another activeagent may block IL-1β receptors, e.g., an interleukin-1 receptorantagonist protein or peptide that blocks the interaction between IL-1βand the IL-1β receptor.

Yet another active agent modifies the release of IL-1β or an antibody toan IL-1β to reduce or neutralize IL-1β, or even an IL-1β receptor. Inone specific example, the active agent may be a naturally occurring IL-1receptor antagonist provided at between about 1, 10, 15, 25, 50, 100,125, 150, 175, 200 and even 250 mg per day. Often doses may be halvedfor pediatric patients. The active agent may also be a single-strandedRNA molecule, a double-stranded RNA molecule, an antisense RNA molecule,a small inhibitory RNA (siRNA) molecule, or an inhibitory RNA molecule(RNAi) designed to target and reduce the expression of interleukin-1βreceptor. Examples of active agents that affect or inhibit the releaseof IL-1β include anion transport inhibitors, e.g., lipoxins, andalpha-tocopherol. Other examples include opioids that inhibit aproteolytic enzyme that converts the inactive IL-1β precursor to itsmature, active form. Yet other examples include antibodies thatneutralize the biological function of IL-1β.

Yet another example of the present invention is a method for treating apatient with systemic onset juvenile idiopathic arthritis byadministering to the patient a composition having an effective amount ofat least one agent capable of reducing or blocking the bioavailabilityof an interleukin-1β. The composition may include an effective amount ofat least one agent capable of reducing or blocking the bioavailabilityof an interleukin-1β receptor. For example, a composition for treatingSOJIA includes a therapeutically effective amount of one or more activeagents adapted for delivery to a joint that reduces the inflammation ofthe joint affected with SOJIA. The composition helps reduce one or moreof the following symptoms: fever, rash, arthritis and white blood cellcount of a SOJIA patient. The composition may include one or more activeagents that are: modifiers of interleukin-1 beta (IL-1β) genetranscription; modifiers of IL-1β gene translation; siRNAs that targetthe expression of IL-1β; IL-1β receptors blockers; interleukin-1receptor antagonist proteins; interleukin-1 receptor antagonistpeptides; active agents that modify the release of IL-1β; antibodiesthat neutralize IL-1β; antibodies that blocks an IL-1β receptor;recombinant, naturally occurring IL-1 receptor antagonists; aniontransport inhibitors, lipoxins and alpha-tocopherol that inhibit therelease of IL-1β; opioids that inhibits a proteolytic enzyme thatconverts the inactive IL-1β precursor to its mature, active form;antibodies that neutralizes the biological function of IL-1β, andmixtures or combinations thereof. The pharmaceutical formulation may beadapted to treat joint autoinflammation and may be a made available in avariety of dosage forms, including, intravenous, intramuscular, oral,nasal, rectal, peritoneal, sub-cutaneous, intra-cutaneous (e.g., apatch), and the like.

The present invention also include a method of monitoring a therapeuticresponse to an autoinflammatory diease, e.g., SOJIA by monitoring thefever, rash, arthritis, white blood cell count and/or the sedimentationrate of white blood cells after treatment using the methods andcompositions taught and disclosed herein. The present invention willfind utility in a wide variety of contexts, e.g., treating an animalsuffering from an autoinflammation by administering a therapeutic agentto an animal suffering from autoinflammation, wherein that agentincluding an antagonist of Interleukin 1β function in a pharmaceuticallyacceptable carrier, wherein the antagonist suppresses theautoinflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a graphic rendering of gene array data that depicts genesupregulated (red), downregulated (blue) or unchanged (yellow) uponincubation of PBMCs from four healthy volunteers with either autologousserum (AS) or the sera from three SOJIA patients;

FIG. 2A is a graph that compares the induction of Interleukin 1-Beta(IL-b) protein secretion in healthy PBMCs incubated with SOJIA sera;

FIG. 2B is a graph that compares the induction of IL-1b proteinsecretion in SOJIA patients with and without systemic symptoms; and

FIGS. 3A through F are graphs that shows the values of temperature (FIG.3A); active joint count (FIG. 3B); WBC (FIG. 3C); hemoglobin (FIG. 3D);platelet count (FIG. 3E); and ESR in 9 SOJIA patients (FIG. 3F).

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. As used in the claim(s),in conjunction with the word “comprising,” the words “a” or “an” maymean one or more than one. As used herein, “another” may mean at least asecond or more of an item. The terminology herein is used to describespecific embodiments of the invention, but their usage does not delimitthe invention, except as outlined in the claims.

As used herein, the term the “therapeutically effective” is used todefine the amount of a compound required to improve some symptomassociated with autoinflammatory disease, e.g., SOJIA. For example, inthe treatment of autoinflammation, the present invention includes one ormore active agents that improve the health or movement of a joint to anydegree or arrests any symptom of a joint disease would betherapeutically effective. A therapeutically effective amount of acompound is not required to cure a disease but will provide a treatmentfor a disease.

As used herein, the phrase “a therapeutically effective amount of one ormore active agents that reduces the bioavailability of interleukin-1β”is the amount of an inhibitor of IL-1β expression, translation,processing, release or activity; an inhibitor of IL-1β receptorexpression, translation, processing, release or activity; and/orinhibitors of second messenger(s) expression, translation, processing,release or activity that are downstream from the IL-1β receptor whenadministered at, to or about a joint, or to an animal systemically orlocally, that reduced the scope and extent of autoinflammation.

As used herein the term, “interleukin-1β receptor” is used to describethe cognate receptor for interleukin-1β. Non-limiting examples of agentsthat may specifically bind to, whether causing activation of thereceptor or not, include: interleukin-1β, anti-interleukin-1β receptorantibodies or fragments thereof, peptides, proteins, glycoproteins,lipoproteins, epitopes, lipids, lectins, carbohydrates, multi-molecularstructures, and specific conformation of one or more molecules. A“receptor” is a naturally occurring molecule or complex of moleculesthat is generally present on the surface of cells of a target organ,tissue or cell type, e.g., a joint.

As used herein the term “modify” or “modifies” is meant to include up ordown regulation of the function of a gene or gene product, e.g.,affecting the transcription, translation, processing, release ormodification of a gene or gene product. The active agent may modify thetranscription of the interleukin-1β gene. Examples of modificationinclude, e.g., transcriptional or post-transcriptional silencing,changes to message stability and the like. Examples ofpost-translational modifications include maturation of the gene productor protein, post-translational modifications (e.g., glycosylation,di-sulfide bonding, myristylation, protease cleavage, association withother proteins, ubiquitination, etc.). The processing, transport andrelease of the protein may also be modified, e.g., by placing in storageorganelles prior to release, by association with other proteins thataffect release and the like.

The present inventors have recognized that SOJIA is an autoinflammatorydisease rather than an autoimmune disease. Therefore, one aspect of thepresent invention is treatment of SOJIA aimed at targeting the cells(and the cell products) of the innate immune system rather than theadaptive immune system.

According to the present invention, IL-1β has now been found to be afundamental mediator of inflammation in “autoinflammatory” diseases.Moreover, IL-1β modulation can be effective in treatment ofautoinflammatory diseases including but not limited to SOJIA.

In one embodiment of the invention the formulation will include atherapeutically effective amount of one or more IL-1 inhibitors, such astype I or type II IL-1 receptor (e.g., IL-1 binding fragments of type IIIL-1 receptor, see e.g., U.S. Pat. No. 5,350,683, relevant portionsincorporated herein by reference); IL-1 binding and IL-1 inhibitoryfragments of type I IL-1 receptor; IL-1 receptor antagonist, IL-1 betaconverting enzyme (ICE) inhibitors, antibodies to IL-1, including IL-1alpha and IL-1 beta and other IL-1 family members, and therapeuticsknown as IL-1 traps and antagonistic type I IL-1 receptor antibodies.Other specific IL-1 inhibitors for use with the present inventioninclude IL-I RA and variants or fragments thereof (see e.g., U.S. Pat.No. 5,922,573, relevant portions incorporated herein by reference). Yetanother active agent is an IL-1 beta converting enzyme (ICE) inhibitor,e.g., peptidyl and small molecule ICE inhibitors including thosedescribed in PCT patent application WO 93/16710; and European patentapplication 0 547 699, relevant portions incorporated herein byreference). Non-peptidyl compounds (see e.g., U.S. Pat. No. 6,121,266,relevant portions incorporated herein by reference) and ICE (see e.g.,U.S. Pat. No. 6,204,261, relevant portions incorporated herein byreference). Other IL-1 antagonists include chimeric proteins thatinclude portions of both an antibody molecule and an IL-1 antagonistmolecule, chimeras and multimers thereof, and IL-1 antagonists such aspeptides derived from IL-1 that are capable of binding competitively tothe IL-1 signaling receptor, IL-1 RI and/or IL-1 RI.

In particular, it has now been found that administering an agent tosignificantly reduce or effectively block the bioavailability ofinterleukin-1 beta (IL-1β) is a therapeutic option for SOJIA patients.As used herein, significant reduction of the bioavailability of IL-1β isdefined as a sufficient reduction of bioavailable IL-1β to effect areduction in symptoms and/or disease progression in SOJIA patients. Inthe present invention, at least one agent capable of significantlyreducing or effectively blocking the bioavailability of IL-1β isadministered to a patient diagnosed with SOJIA to effect a reduction insymptoms and disease progression. It is contemplated that more than oneagent capable of reducing or blocking the bioavailability of (IL-1β) canbe administered to an SOJIA patient either concomitantly orsequentially. It is further contemplated that one or more agents capableof reducing or blocking the bioavailability of (IL-1β) can beadministered to an SOJIA patient in combination with other drugtreatments either concomitantly or sequentially.

Techniques and compositions for making useful dosage forms using thepresent invention are described in one or more of the followingreferences: United States Pharmacopeia (USP #24 NF19) (Hardcover Textw/3 Supplements)(United States Pharmacopeial 2002). Remington: TheScience and Practice of Pharmacy, (21st Edition, formerly, Remington'sPharmaceutical Sciences), Mack Publishing Co, 2005); Advances inPharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992), andthe like, relevant portions incorporated herein by reference.

Generally, the therapeutic formulations of the present invention will beprovided to a person in need of preventive or therapeutic interventionin water, a suitable oil, saline, aqueous dextrose (e.g., glucose,lactose and related sugar solutions) and glycols (e.g., propylene glycolor polyethylene glycols) or other suitable carriers for intravenous orparenteral administration. Solutions for parenteral administrationinclude generally, a water soluble salt of the active ingredient,suitable stabilizing agents, and if necessary, buffering salts. Citricacid and its salts and/or sodium EDTA may also be included to increasestability. Antioxidizing agents, e.g., sodium bisulfite, sodium sulfiteand/or ascorbic acid, either alone or in combination, are suitablestabilizing agents. In addition, the solutions may includepharmaceutically acceptable preservatives, e.g., benzalkonium chloride,methyl- or propyl-paraben, and/or chlorobutanol. Suitable pharmaceuticalcarriers are described in Remington's (supra).

In one embodiment, treatment of SOJIA includes administering an amountof at least one agent to significantly reduce or effectively block thetranscription of the interleukin-1 beta (IL-1β) gene. Exemplary agentsfor the present invention suitable for blocking the transcription of theinterleukin-1 beta (IL-1β) gene include but are not limited toanti-sense RNA compounds (Mehta R C, et al. 2000. “Intercellularadhesion molecule-1 suppression in skin by topical delivery ofanti-sense oligonucleotides,” J Invest Dermatol 115):805-812; Frankel AE, et al. 2001. “Novel therapeutics for chemotherapy-resistant acutemyeloid leukaemia,” BioDrugs 15:55-57; Lorenz H M and Kalden J R. 2001.“New therapy developments in rheumatoid arthritis,” Z Rheumatol60:326-32.; Ideo G and Bellobuono A. 2002. “New therapies for thetreatment of chronic hepatitis C,” Curr Pharm Des 8:959-966; Sandborn WJ and Targan S R. 2002. “Biologic therapy of inflammatory boweldisease,” Gastroenterology 122:1592-1608).

In another embodiment, treatment of SOJIA includes administering anamount of at least one agent to significantly reduce or effectivelyblock IL-1β receptors, including interleukin-1 receptor antagonistproteins or peptides. Exemplary agents for the present inventionsuitable for block IL-1β receptors include but are not limited to anisolated naturally occurring IL-1 receptor antagonist. One such agent iscommercially available Anakinra (Kineret®, made by Amgen), a recombinantform of the naturally occurring IL-1 receptor antagonist.

In yet another embodiment, treatment of SOJIA includes administering aneffective amount of at least one agent to significantly reduce oreffectively block the release of IL-1β. Exemplary agents include aniontransport inhibitors, lipoxins, and alpha-tocopherol. In yet anotherembodiment, treatment of SOJIA includes administering an effectiveamount of at least one agent to significantly reduce or effectivelyblock the activity of IL-1β converting enzyme, a proteolytic enzyme thatconverts the inactive IL-1β precursor to its mature, active form.Exemplary agents include morphine and other opioids.

It is further contemplated that the present invention includes theadministration of an antibody to IL-1β. The antibody to IL-1β can bemade by any means known in the art. For example, monoclonal antibodiescan be generated by immunizing mice with recombinant IL-1β or one orseveral synthetic peptides including epitopes that are regarded ashighly antigenic. Alternatively, polyclonal antibodies can also beraised by immunizing rabbits using the above described antigens. Thegenerated antibodies are examined to determine their ability to interactwith IL-1 protein and their ability to block IL-1 mediated cellularresponses. Once generated using hybridoma technology, the monoclonalantibodies are then humanized by swapping mouse and human framework andconstant regions. According to the present invention, antibodies thatblock the biological effect of IL-1β block the IL-1-induced release ofIL-8 from fibroblasts as described by Kaplanski G et al. 1994.“Interleukin-1 induces interleukin-8 secretion from endothelial cells bya juxtacrine mechanism,” Blood 84:4242-4248. The IL-1β antibodies arethen administered intravenously or subcutaneously to SOJIA patients inan effective amount to significantly reduce or effectively neutralizethe biological function of IL-1β.

According to the present invention, the therapeutic response of SOJIApatients is monitored by assessing the patient's clinical improvement,i.e., disappearance and/or improvement of the fever, rash and arthritis,as well as normalization of the white blood cell count and thesedimentation rate.

Based on the finding that culturing SOJIA patient's sera with healthyblood mononuclear cells (PBMCs) in vitro results in significantinduction of IL-1 beta (IL-1β) transcription (Example 1), two patientswith persistently active disease for over 1 year in spite of moreconventional aggressive treatment were treated with a commerciallyavailable anti-IL-1 medication (Anakinra, Amgen Inc., Thousand Oaks,Calif.), known to neutralize the biological activity of IL-1. Asillustrated in Example 2 and 3, both patients became asymptomatic theday after initiation of therapy and remained asymptomatic for over 3months.

EXAMPLE 1

Incubation of healthy PBMCs with SOJIA serum induces the transcriptionalupregulation of Interleukin 1β. The gene expression profile was analyzedfor healthy donor PBMCs before and after incubation with autologousserum or serum from two untreated and two treated SOJIA patients for sixhours in vitro. After incubation with the serum, the PBMCs wereprocessed to extract the RNA, and the RNA was hybridized to AffymetrixU133 A and B microarray chips. In this way, a wide array of genes whosetranscription would be up-regulated or down-regulated with the SOJIAserum was screened.

Patient Population. PBMCs and sera from 23 SOJIA patients (15 females, 8males, average age 7.1 years) fulfilling the ACR diagnostic criteria³⁴were collected on repeated occasions. Patients were classified as activeif they had systemic symptoms (fever and/or rash) and/or activearthritis (swollen and/or tender and limited joints). The controlpopulation consisted of 12 children (average 14 years) and 7 adults(average 35 years). The sera from healthy controls were cultured onrepeated occasions with autologous and heterologous PBMCs. Patients andpediatric controls were recruited at Texas Scottish Rite Hospital forChildren in Dallas. The study was approved by the Institutional ReviewBoards of UT Southwestern Medical Center, Texas Scottish Rite Hospitaland Baylor Health Care System (IRB #0199017, 0701-513) and informedconsent was obtained from parents or legal guardians.

PBMC cultures and RNA extraction. PBMCs were obtained byFicoll-Histopaque gradient centrifugation of 20 ml of blood. PBMCs werecultured (10⁶/ml) in RPMI 1640 supplemented with 20% autologous or SOJIApatient's serum for 6 hrs. RNA was extracted using RNAeasy kit (Qiagen,Valencia, Calif.) and assessed using an Agilent 2100 Bioanalyzer(Agilent, Palo Alto, Calif.). Supernatants were frozen at −80° C. PBMCswere cultured with RPMI 1640 and 10% fetal calf serum with PMA (50ng/ml)-lonomycin (1 μg/ml) for 24 hrs. Cells were harvested and the RNAextracted as above. Supernatants were frozen at −80° C.

Microarrays and Real-time PCR. Samples for microarray analysis wereprocessed as described¹⁸ and hybridized to the HG U133A AffymetrixGeneChip array containing 22,283 probe sets (Affymetrix Inc., SantaClara, Calif.) at 45° C. for 16 hours. GeneChip arrays were washedstained and scanned according to protocols described in the GeneChipExpression Analysis Technical Manual (Affymetrix). Scanned GeneChipswere visually inspected for abnormalities or irregularities.

FIG. 1 is a depiction of a gene array for cytokine-cytokine expression.Incubation of healthy PBMCs with autologous serum (AS) or with the serumfrom 4 SOJIA patients for 6 hr in vitro. SOJIA serum was found to inducethe upregulation of transcription of members of the IL-1 family.Asterisk indicates that gene transcription is significantly upregulatedin vivo in SOJIA patients PBMCs.

Data Analysis. Intensity values were scaled to 500 using global scalingin MAS 5.0 and data were exported in MS Excel for import into GeneSpringsoftware (Silicon Genetics, Redwood City, Calif.) for gene expressionanalyses. No “per chip” normalization was performed as global scalinghad been applied in MAS 5.0. Global scaling adjusts for chip-to chipvariations in hybridization intensities. Subsequent samples werenormalized to pediatric healthy controls and/or to the median of allsamples. Statistical comparisons were performed in GeneSpring using bothparametric (Welch's approximate t-test) and non-parametric (Mann-WhitneyU-test) methods. Unsupervised hierarchical clustering was performed tovisualize transcripts that had a control signal of 50 or above (abovethe background intensity) and that were identified as “present”according to MAS 5.0 in 15% of all samples. Statistical comparisons wereperformed in GeneSpring using both parametric (Welch's approximatet-test) and non-parametric (Mann-Whitney U-test) methods. Unsupervisedhierarchical clustering was performed to visualize transcript/samplerelationships using standard correlation, Pearson correlation orEuclidian distance where indicated.

Two-step RT-PCR was performed using Applied Biosystems TaqMan Assays onDemand probe and primer sets according to the manufacturer'sinstructions and the ABI Prism 7700 Sequence detection System (AppliedBiosystems). The endogenous GAPDH gene and/or 18S RNA were used forcorrecting the results with the comparative threshold cycle (C_(T))method for relative quantification as described by the manufacturer.

Multiplex Analysis (Luminex). Culture supernatants were analyzed for 6cytokines and chemokines using the FluorikineMAP cytokine assay kit (R&DSystems, Minneapolis, Minn.) as per manufacturer's protocol.

FIG. 2A is a graph that compares the induction of Interleukin 1-Beta(IL-b) protein secretion in healthy PBMCs incubated with SOJIA sera.Supernatants from 6 hr incubation of healthy PBMCs with 12 SOJIA seraand 3 healthy controls were assayed for IL-1b production by Luminex.

FIG. 2B is a graph that compares the induction of IL-1b proteinsecretion in SOJIA patients with and without systemic symptoms. IL-1bprotein secretion is preferentially induced by sera from SOJIA patientsexperiencing systemic symptoms (SOJIA1, n=5) over those patients withonly active arthritis (SOJIA2, n=7). All results were analyzed usingnon-parametric tests (Mann-Whitney).

FIGS. 3A through F are graphs that shows the values of temperature (FIG.3A); active joint count (FIG. 3B); WBC (FIG. 3C); hemoglobin (FIG. 3D);platelet count (FIG. 3E); and ESR in 9 SOJIA patients (FIG. 3F). Xvalues represent months prior (-2) to starting of Anakinra treatment (0)and up to 2-12 months of follow up (average 6.6. months). Arrowsindicate the time of treatment initiation. P values were calculated attime 0 and at 2 month follow-up (paired, two-tailed t test).

EXAMPLE 2

Interleukin 1β treatment of SOJIA patient. A 17 year old Hispanic femalediagnosed with SOJIA at the age of 15 was treated according to themethod of the present invention. She presented with persistent (>2month) daily fever, a generalized pruritic rash and joint pain. On exam,she had swelling and limitation of the radio-carpal andcarpal-metacarpal joints bilaterally. Her laboratory tests revealed highwhite blood cell count (WBC), low hemoglobin (Hgb), high platelets(Plts) and elevated erythrocyte sedimentation rate (ESR). An extensiveinfectious disease work up yielded negative results. The patientreceived oral and IV high dose methyl prednisolone and eventually wasplaced on methotrexate. Her symptoms improved, and the patient wasasymptomatic for several months during which the steroid treatment wasslowly tapered. A few months later the symptoms recurred. Steroids werereinitiated without success. Anti-TNF IV therapy (Infliximab) was triedat a dose of 5 mg/kg every month for approximately 6 months without anysignificant effect. After the patient had been symptomatic for over oneyear, IL-1 antagonist was administered in the form of Anakinra, 100 mg(1 cc) as a subcutaneous daily injection. The day after the firstsubcutaneous injection, the patient was asymptomatic, with thedisappearance of fever, rash and joint swelling. Two follow-up visitsconfirmed that she remained asymptomatic at 2 and 3 monthspost-initiation of therapy. Laboratory tests also reflected thisresponse, as WBC, Hgb, Plt number and ESR were within normal limits.

EXAMPLE 3

Interleukin 1β treatment of SOJIA patient. A 9 year old Hispanic malewas diagnosed with SOJIA at the age of 7, when he presented withprolonged daily spiking fever, rash, pericarditis and joint and musclepain. Laboratory tests showed high WBC, low Hgb, high platelets andelevated ESR. Bone marrow analysis and an extensive infectious work upfailed to yield a specific diagnosis. The patient was treated with highdose IV methylprednisolone, and symptoms improved. Over the next twoyears, the patient presented flares and remissions. The last flare didnot respond to high dose steroids, and the patient developed swellingover the radio-carpal joints bilaterally. Therapy with IL-1 antagonistAnakinra was initiated at a dose of 50 mg (0.5 cc) as a subcutaneousdaily injection. The day after the first subcutaneous injection, thepatient became asymptomatic, with the disappearance of fever, rash,joint pain and swelling, and he remained free of symptoms for over 3months.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A method for treating a joint with systemic onset juvenile idiopathicarthritis comprising administering to the joint a composition comprisinga therapeutically effective amount of one or more active agents thatreduces the bioavailability of interleukin-1β, wherein the active agentcomprises an antibody to an IL-1β to reduce or neutralize IL-1β. 2-14.(canceled)
 15. The method of claim 1, wherein the active agent comprisesan antibody that neutralizes the biological function of IL-1β.
 16. Amethod for treating a patient with systemic onset juvenile idiopathicarthritis comprising administering to the patient a compositioncomprising an effective amount of at least one antibody to an IL-1βcapable of reducing or blocking the bioavailability of aninterleukin-1β.
 17. A method for treating a patient with systemic onsetjuvenile idiopathic arthritis comprising administering to the patient acomposition comprising an effective amount of at least one antibodycapable of reducing or blocking the bioavailability of an interleukin-1βreceptor. 18-23. (canceled)
 24. A method of treating an animal sufferingfrom an autoinflammation comprising administering a therapeutic agent toan animal suffering from autoinflammation, wherein that agent comprisesan antagonist of Interleukin 1β function in a pharmaceuticallyacceptable carrier, wherein the antagonist comprises an antibody to anIL-1β to reduce or neutralize IL-1β and suppresses the autoinflammation.25.